1. Field of Invention
The present invention relates to a novel cell pattern recovery tool (or a cell cultivation support for cell pattern recovery). More specifically, the present invention relates to a cell pattern recovery tool that can cultivate cells so as to have a desired cell shape to form a cell pattern (sheet) that can be rapidly recovered. Further, the present invention relates to a process for producing the cell pattern recovery tool and a process for preparing a cell pattern using the cell pattern recovery tool.
2. Background Art
A cell sheet is a sheet-like cell aggregate comprising cells that have been linked to one another through at least a single layer by cell junction. The cell pattern is a cell sheet that has been formed so as to have a desired specific shape. The cell sheet and the cell pattern are often used, for example, for regenerative medical techniques.
The cell sheet is obtained by cell cultivation on a support such as a petri dish. The cell sheet formed on the support is strongly bound to the surface of the support, for example, through adhesive molecules. Accordingly, rapidly separating the cell sheet from the cultivation support without breaking binding between cells is not easy. Accordingly, in the case of a cell pattern, due to a specific shape, further difficulties are encountered in rapidly, stably, and reliably separating the cell pattern from the cultivation support.
Up to now, various studies have been made to propose methods that can efficiently separate a cell sheet from a cell cultivation support. Conventional separating methods are classified roughly into two groups. The methods belonging to the first group are to weaken binding between the support and the cells by an enzymatic reaction. The methods belonging to the second group are to use a support having low cell adherence or a support with adherence of cells thereto being variable.
More specifically, the methods belonging to the first group comprise degrading proteins constituting intercellular adhesion molecules (tight junction, adhering junction, desmosome junction, gap junction, or hemidesmosome junction), collagen connective tissues surrounding a culture, and extracellular matrix (ECM) formed between cells and the support by enzymes such as proteases (proteolytic enzymes) and collagenases (collagenolytic enzymes). These methods, however, weaken junction between cells and the surface of the support, as well as junction between cells. Accordingly, the methods belonging to the first group cause not a little damage to the cell sheet. The binding substances degraded by this method are substances produced in cells which are cultivated, tissues, and organs. Accordingly, even after the separation, binding substances that have been degraded under given conditions and in a given period can be regenerated. The regeneration of the binding substances, however, takes a lot of time.
Regarding a method using a support with adherence of cells thereto being variable among the methods belonging to the second group, for example, Japanese Patent Publication No. 104061/1994 (EP 0382214A) (document 1) discloses a support having a cell growth surface covered with a temperature responsive polymer. This document describes a process for producing a temperature responsive polymer layer by a graft polymerization method in which electron beam is applied to cause both a monomer polymerization reaction and a (grafting) reaction by which at least one end of a temperature responsive polymer is covalently bonded to molecules constituting a base material and thus to immobilize the temperature responsive polymer to the surface of the base material. Further, for example, Japanese Patent Application Laid-Open Publication No. 192130/1993 (document 2) also refers to the use of a temperature responsive polymer in cell cultivation. In these documents, however, no study has been made to rapidly and stably recover a cell pattern in the cultivation support using a temperature responsive polymer.
Japanese Patent Application Laid-Open Publication No. 082119/2003 (document 3) discloses a cell recovery membrane and a process for producing the cell recovery membrane. Further, Japanese Patent Application Laid-Open Publication No. 342112/2005 (US 2007259328A) (document 4) discloses a tissue body which has been formed in vitro and recovered in such a state that a cell pattern is maintained, and a process for producing the tissue body. Japanese Patent Application Laid-Open Publication No. 8975/2006 (document 5) discloses the cultivation of desired cells according to a desired pattern. These documents, however, do not describe that a temperature responsive polymer layer is provided and a cell adhesion inhibiting material layer is formed, on the temperature responsive polymer layer, in a desired cell pattern shape. Accordingly, these documents neither describe nor suggest the formation of a cell pattern which is then rapidly recovered. In the method disclosed in patent document 5, ultraviolet irradiation is also performed for cell pattern separation.
Therefore, a cultivation support, that is, a cell pattern recovery tool, that can rapidly recover a cell pattern while maintaining the cell pattern stably and reliably under minimally invasive conditions for the cells has been still demanded.